Prevalence of deltaF508, G551D, G542X, and R553X mutations among cystic fibrosis patients in the North of Brazil

Cystic fibrosis (CF) is the most common genetic disease among Caucasians and is rare among sub-Saharan Africans. The Brazilian population is not ethnically homogeneous but it is the result of three-way ethnic admixture of Europeans, Africans and Amerindians in varying proportions, depending on the region. In the present study, we investigated 33 patients who had been diagnosed and are currently under treatment for CF at the University Hospital João de Barros Barreto, Belém, Pará State. The molecular analysis for G542X, G551D and R553X mutations was performed by PCR followed by RFLP using BstNI, HincII and MboI, respectively, in polyacrylamide gel eletrophoresis and stained with AgNO3. ThedeltaF508 mutation (a deletion of 3 bp) was only analyzed by polyacrylamide gel electrophoresis and stained with AgNO3. Each sample was analyzed for regions of interest in the CFTR gene using amplified by PCR and specific primers. The deltaF508 and G551D mutations presented frequencies of 22.7 and 3%, respectively. In 74.3% of the remaining patients, none of the mutations investigated was found. The present study characterized in a sample of patients with an established clinical diagnosis of CF (asthma, repeated bronchopneumonia, disorders of nutritional status, etc.) the most frequent mutation ( deltaF508) in the North region of Brazil and is also the first report of the G551D mutation. In spite of the wide spectrum of CF mutations and the heterogeneous ethnic origin of the Amazon population, the molecular diagnosis is a helpful additional tool for the diagnosis and treatment of CF patients.

No evidence of association of MUC-1 genetic polymorphism with embryo implantation failure

Pregnancy loss can be caused by several factors involved in human reproduction. Although up to 50% of cases remain unexplained, it has been postulated that the major cause of failed pregnancy is an error of embryo implantation. Transmembrane mucin-1 (MUC-1) is a glycoprotein expressed on the endometrial cell surface which acts as a barrier to implantation. The gene that codes for this molecule is composed of a polymorphic tandem repeat of 60 nucleotides. Our objective was to determine if MUC-1 genetic polymorphism is associated with implantation failure in patients with a history of recurrent abortion. The study was conducted on 10 women aged 25 to 35 years with no history of successful pregnancy and with a diagnosis of infertility. The control group consisted of 32 patients aged 25 to 35 years who had delivered at least two full-term live children and who had no history of abortions or fetal losses. MUC-1 amplicons were obtained by PCR and observed on agarose and polyacrylamide gel after electrophoresis. Statistical analysis showed no significant difference in the number of MUC-1 variable number of tandem repeats between these groups (P > 0.05). Our results suggest that there is no effect of the polymorphic MUC-1 sequence on the implantation failure. However, the data do not exclude MUC-1 relevance during embryo implantation. The process is related to several associated factors such as the mechanisms of gene expression in the uterus, specific MUC-1 post-translational modifications and appropriate interactions with other molecules during embryo implantation.

Different responses of the GlnB and GlnZ proteins upon in vitro uridylylation by the Azospirillum brasilense GlnD protein

Azospirillum brasilense is a diazotroph found in association with important agricultural crops. In this organism, the regulation of nitrogen fixation by ammonium ions involves several proteins including the uridylyltransferase/uridylyl-removing enzyme, GlnD, which reversibly uridylylates the two PII proteins, GlnB and GlnZ, in response to the concentration of ammonium ions. In the present study, the uridylylation/deuridylylation cycle of A. brasilense GlnB and GlnZ proteins by GlnD was reconstituted in vitro using the purified proteins. The uridylylation assay was analyzed using non-denaturing polyacrylamide gel electrophoresis and fluorescent protein detection. Our results show that the purified A. brasilense GlnB and GlnZ proteins were uridylylated by the purified A. brasilense GlnD protein in a process dependent on ATP and 2-oxoglutarate. The dependence on ATP for uridylylation was similar for both proteins. On the other hand, at micromolar concentration of 2-oxoglutarate (up to 100 µM), GlnB uridylylation was almost twice that of GlnZ, an effect that was not observed at higher concentrations of 2-oxoglutarate (up to 10 mM). Glutamine inhibited uridylylation and stimulated deuridylylation of both GlnB and GlnZ. However, glutamine seemed to inhibit GlnZ uridylylation more efficiently. Our results suggest that the differences in the uridylylation pattern of GlnB and GlnZ might be important for fine-tuning of the signaling pathway of cellular nitrogen status in A. brasilense.

ABCB1 C1236T, G2677T/A and C3435T polymorphisms in systemic lupus erythematosus patients

P-glycoprotein (Pgp), the ABCB1 gene product, acts as an efflux pump that transports a large variety of substrates and is a mechanism of cell protection against xenobiotics. An increasing number of studies have shown that some ABCB1 polymorphisms may affect Pgp expression and activity, as well as affecting the development and susceptibility to diseases and pharmacological response. High activity of Pgp has been detected in systemic lupus erythematosus (SLE) patients. The C1236T, G2677T/A, and C3435T are the most commonly studied single nucleotide polymorphisms in the ABCB1 gene. Therefore, their frequencies were determined in Brazilian individuals with European ancestry (N = 143) and in SLE patients (N = 137). Genotyping was performed by PCR-RFLP analysis using specific primers followed by incubation with the appropriate restriction enzymes. The resulting DNA fragments were visualized on agarose or polyacrylamide gels. No statistically significant differences were observed in allelic and genotypic frequencies between SLE and healthy subjects (Fisher exact test). Nevertheless, the 2677A allelic frequency was lower in SLE patients with malar rash (0.007) compared with patients without this feature (0.04; P = 0.0054), while the frequency of this variant was higher in SLE patients with pleuritis (0.07) compared with patients without this feature (0.01; P = 0.0156). We suggest that although the ABCB1 polymorphisms do not directly interfere in SLE susceptibility, their evaluation, especially the 2677A allele, in other immunological processes may be interesting since they can interfere in clinical features of this disease.

Proteomic analysis of cytosolic proteins associated with petite mutations in Candida glabrata

The incidence of superficial or deep-seated infections due to Candida glabrata has increased markedly, probably because of the low intrinsic susceptibility of this microorganism to azole antifungals and its relatively high propensity to acquire azole resistance. To determine changes in the C. glabrata proteome associated with petite mutations, cytosolic extracts from an azole-resistant petite mutant of C. glabrata induced by exposure to ethidium bromide, and from its azole-susceptible parent isolate were compared by two-dimensional polyacrylamide gel electrophoresis. Proteins of interest were identified by peptide mass fingerprinting or sequence tagging using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer. Tryptic peptides from a total of 160 Coomassie-positive spots were analyzed for each strain. Sixty-five different proteins were identified in the cytosolic extracts of the parent strain and 58 in the petite mutant. Among the proteins identified, 10 were higher in the mutant strain, whereas 23 were lower compared to the parent strain. The results revealed a significant decrease in the enzymes associated with the metabolic rate of mutant cells such as aconitase, transaldolase, and pyruvate kinase, and changes in the levels of specific heat shock proteins. Moreover, transketolase, aconitase and catalase activity measurements decreased significantly in the ethidium bromide-induced petite mutant. These data may be useful for designing experiments to obtain a better understanding of the nuclear response to impairment of mitochondrial function associated with this mutation in C. glabrata.

Effect of castration on renal glycosaminoglycans and their urinary excretion in male and female rats with chronic renal failure

Glycosaminoglycans (GAGs) participate in a variety of processes in the kidney, and evidence suggests that gender-related hormones participate in renal function. The aim of this study was to analyze the relationship of GAGs, gender, and proteinuria in male and female rats with chronic renal failure (CRF). GAGs were analyzed in total kidney tissue and 24-h urine of castrated (c), male (M), and female (F) Wistar control (C) rats (CM, CMc, CF, CFc) and after 30 days of CRF induced by 5/6 nephrectomy (CRFM, CRFMc, CRFF, CRFFc). Total GAG quantification and composition were determined using agarose and polyacrylamide gel electrophoresis, respectively. Renal GAGs were higher in CF compared to CM. CRFM presented an increase in renal GAGs, heparan sulfate (HS), and proteinuria, while castration reduced these parameters. However, CRFF and CRFFc groups showed a decrease in renal GAGs concomitant with an increase in proteinuria. Our results suggest that, in CRFM, sex hormones quantitatively alter GAGs, mainly HS, and possibly the glomerular filtration barrier, leading to proteinuria. The lack of this response in CRFMc, where HS did not increase, corroborates this theory. This pattern was not observed in females. Further studies of CRF are needed to clarify gender-dependent differences in HS synthesis.

Changes in epidemiology of rotavirus in the Triângulo Mineiro region of Brazil: lack of two consecutive rotavirus seasons

Rotaviruses are the main cause of infantile acute diarrhea, and a monovalent (G1P[8]) vaccine against the virus was introduced into the Brazilian National Immunization Program for all infants in March 2006. The objectives of this study were to determine the rate and genotype distribution of rotavirus causing infantile diarrhea in the Triângulo Mineiro region of Brazil during 2011-2012 and to assess the impact of local vaccination. Fecal specimens were analyzed for detection and characterization of rotavirus using polyacrylamide gel electrophoresis, reverse transcription followed by polymerase chain reaction (PCR), and PCR-genotyping assays. Overall, rotavirus was diagnosed in 1.7% (6/348) of cases. Rotavirus positivity rates decreased 88% [95% confidence intervals (CI)=15.2, 98.3%; P=0.026] in 2011 and 78% (95%CI=30.6, 93.0%; P=0.007) in 2012 when compared with available data for baseline years (2005/2006) in Uberaba. In Uberlândia, reductions of 95.3% (95%CI=66.0, 99.4%; P=0.002) in 2011, and 94.2% (95%CI=56.4, 99.2%; P=0.004) in 2012 were also observed compared with data for 2008. The circulation of rotavirus G2P[4] strains decreased during the period under study, and strains related to the P[8] genotype reemerged in the region. This study showed a marked and sustained reduction of rotavirus-related cases, with a lack of rotavirus in the 2011 and 2012 seasons, suggesting a positive impact of the vaccination program.

Electrophoretic analisys to detect and quantify additional whey in milk and dairy beverages

Polyacrylamide gel electrophoresis, SDS-PAGE system, was adjusted to detect the presence of additional whey in dairy beverages distributed in a Brazilian Government School Meals Program. Aqueous solutions of samples in 8 M urea were submitted to a polyacrylamide gel gradient (10% to 18%). Gel scans from electrophoresis patterns of previously adulterated milk samples showed that caseins peak areas decreased while peak areas of beta -lactoglobulin plus alpha -lactalbumin increased as the percentage of raw milk powder replaced by whey powder increased. The relative densitometer areas of caseins or beta -lactoglobulin plus alpha -lactalbumin plotted against the percentage of whey added to the raw milk showed a linear correlation coefficient square higher than 0.97. The caseins plot was used to determine the percentage of additional whey in 116 dairy beverages, chocolate or coffee flavor. Considering that the lowest relative caseins concentration found in commercial milk powder samples by the present method was 72%, the dairy beverages containing caseins percentages equal to or higher than this value were considered free of additional whey. Based on this criterion, about 49% of the coffee-flavor dairy beverages and 29% of the chocolate-flavor beverages, among all the samples analyzed were adulterated with whey protein to reach the total protein contents specified on their labels. The present method showed a sensitivity of 5% to additional whey.

Partial purification and characterization of a bacteriocin produced by Enterococcus faecium 130 isolated from mozzarella cheese

Lactic acid bacteria are important in foods as potential probiotics and also due to the ability to produce antimicrobial compounds that can contribute for biopreservation. In this work, the bacteriocin produced by the food isolate Enterococcus faecium 130 was partially purified and characterized. The compound was active against Gram-positive bacteria, including Listeria monocytogenes. It was produced after 4 days of storage at a broad temperature range (4 to 37 °C); it was stable at pH ranging from 2 to 10 with no loss of activity after heating at 100 °C for 15 minutes. Bacteriocin was partially purified by the adsorption-desorption technique, and the analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a molecular mass of 3.5 to 6.5 kDa. These data encourage studies on application of this bacteriocin in food systems as an additional hurdle to microbial growth.

Potential of quixaba (Sideroxylon obtusifolium) latex as a milk-clotting agent

There are several obstacles to the use of chymosin in cheese production. Consequently, plant proteases have been studied as possible rennet substitutes, but most of these enzymes are unsuitable for the manufacture of cheese. The aim of this study was to evaluate the potential of latex from Sideroxylon obtusifolium as a source of milk-clotting proteases and to partially characterize the enzyme. The enzyme extract showed high protease and coagulant activities, with an optimal pH of 8.0 and temperature of 55 °C. The enzyme was stable in wide ranges of temperature and pH. Its activity was not affected by any metal ions tested; but was inhibited by phenylmethanesulfonyl fluoride and pepstatin. For the coagulant activity, the optimal concentration of CaCl2 was 10 µmol L- 1. Polyacrylamide gel electrophoresis showed four bands, with molecular weights between 17 and 64 kDa. These results indicate that the enzyme can be applied to the cheese industry.

Effect of high and low molecular weight glutenin subunits, and subunits of gliadin on physicochemical parameters of different wheat genotypes

Identification of functional properties of wheat flour by specific tests allows genotypes with appropriate characteristics to be selected for specific industrial uses. The objective of wheat breeding programs is to improve the quality of germplasm bank in order to be able to develop wheat with suitable gluten strength and extensibility for bread making. The aim of this study was to evaluate 16 wheat genotypes by correlating both glutenin subunits of high and low molecular weight and gliadin subunits with the physicochemical characteristics of the grain. Protein content, sedimentation volume, sedimentation index, and falling number values were analyzed after the grains were milled. Hectoliter weight and mass of 1000 seeds were also determined. The glutenin and gliadin subunits were separated using polyacrylamide gel in the presence of sodium dodecyl sulfate. The data were evaluated using variance analysis, Pearson's correlation, principal component analysis, and cluster analysis. The IPR 85, IPR Catuara TM, T 091015, and T 091069 genotypes stood out from the others, which indicate their possibly superior grain quality with higher sedimentation volume, higher sedimentation index, and higher mass of 1000 seeds; these genotypes possessed the subunits 1 (Glu-A1), 5 + 10 (Glu-D1), c (Glu-A3), and b (Glu-B3), with exception of T 091069 genotype that possessed the g allele instead of b in the Glu-B3.

Peptide separation of commercial fermented milk during refrigerated storage

Milk is an important source of bioactive compounds. Many of these compounds are released during fermentation and refrigerated storage. The aim of this study was to determine the release of peptides by lactic acid bacteria in commercial fermented milk during refrigerated storage. The size and profile of peptides were analyzed by polyacrylamide gel electrophoresis and sizeexclusion HPLC. During electrophoresis, it was observed that the peptides were released from caseins, whereas β-lactoglobulin was the whey protein with the highest degradation. HPLC analysis confirmed the pattern of peptide formation observed in electrophoresis. Two fractions lower than 2 kDa with aromatic amino acids in their structure were separated. These results were consistent with those reported for structures of peptides with antihypertensive activity. Therefore, the presence of aromatic amino acids in the peptide fractions obtained increases the likelihood of finding peptides with such activity in refrigerated commercial fermented milk. In conclusion, during cold storage, peptides with different molecular weights are released and accumulated. This could be due to the action of proteinases and peptidases of the proteolytic system in lactic acid bacteria.

Accelerated aging of ipê seeds under controlled conditions of storage

This research was aimed at studying effects of storage and accelerated aging on germination and profile of storage proteins in Handroanthus albus seeds. These were stored into a cold chamber (± 8 ºC; RH ± 40%) and after periods of 0, 3, 6, 9, and 12 months of storage, were subjected to accelerated aging for 0, 24, 48, 72, and 96 hours. Relationships between germination and proteins profile were assessed. Germination test was performed at 25 ºC, under constant light. For protein extraction, 125 mg of seeds were macerated in 2 mL of extraction buffer (1M Tris-HCl; pH 8.8) and applied to SDS-PAGE polyacrylamide gel at 80 V .15 h-1. Twelve month storage, combined with 72 hours accelerated aging have increased germination in approximately 65% when compared to non-aged seeds or to seeds with 24 h of accelerated aging. Besides beneficial effects, degradation and synthesis of different proteins were observed. It was concluded that germination of Handroanthus albus seeds, when not subjected to accelerated aging, is favored by storage in cold chamber during three to six months, or from nine to 12 months when subjected to accelerated aging process. Storage proteins may be associated to those increases, and hence further studies are needed.

Characterization and protein fingerprinting of Botrytis cinerea isolates /

Botrytis cinerea isolates collected from Niagara region were treated with different concentrations of the fiingicide, iprodione to test their sensitivity to this fungicide. These Botrytis cinerea isolates were divided into two groups according to their sensitivity to iprodione. Those isolates whose growth was inhibited by iprodione at concentrations < 2|i,g/nil were classified as sensitive isolates. Isolates that were able to show considerable growth at 2|j,g/ml iprodione were classified as resistant isolates. Resistant and sensitive isolates were compared for their morphological and growth characteristics, conidial germination, virulence on grape berries and protein banding profiles. The fungicide iprodione at a concentration of 2|xg/nil inhibited mycelial growth, sporulation and conidial germination of sensitive isolates but not those of resistant isolates. The inhibitory effect of the fungicide was greater on mycelial growth than on conidia germination of the sensitive isolates. Sensitive isolates produced no sclerotia whereas resistant isolates produced large number of sclerotia. The fungicide iprodione affected sclerotial production in the resistant isolates. The number of sclerotia was decreased by the increase of iprodione in the medium. Sporulation of resistant isolates was improved significantly in the presence of iprodione. The resistant isolates were as virulent as the sensitive isolates on grape berries. The sensitive and resistant isolates showed similar protein banding profiles in the absence of iprodione in polyacrylamide gel electrophoresis studies. Similar protein profiles were also observed when these isolates were grown in the presence of low iprodione concentration (0.5|ig/nil). However, in the presence of concentration (0.5|ig/nil). However, in the presence of iprodione at concentration of 5|Xg/nil, one protein band with approximate molecular weight of 83 KDa was present in the growing resistant isolates (and the controls) but was missing in the inhibited sensitive isolates.

Involvement of fimbriae in host-mycoparasite recognition

Extracellular, non-flagellar appendages, termed fimbriae are widespread among fungi. Fungal fimbriae range in diameter from 6-10 nm and exhibit lengths of up to 30 ~m. Fungal fimbriae have been implicated in several functions: adhesion, conjugation and flocculation. A possible role of fimbriae in host-mycoparasite interactions was the focus of this study . Using electron microscopy, fimbriae were observed on the surfaces of Mortiere lla cande labrum, Mortie re lla pusi lla and Phascolomyces articulosus with diameter means of 9.1±0.4 nm, 9.4±0.5 nm and 8.6±0.6 nm, respectively, and lengths of up to 25 ~m. Fimbriae were not observed on the surface of the mycoparasite, Piptocephalis virginiana. Polyclonal antiserum (AU) prepared against the fimbrial protein of Ustilago violacea cross-reacted with 60 and 57 kDa M. candelabrum proteins. In addition, AU cross-reacted with 64 kDa proteins from both M. pusilla and P. articulosus. The proteins that cross-reacted with AU were electroeluted from polyacrylamide gels and were shown to subsequently form fibrils. The diameter means for the electroeluted fibrils were: for M. candelabrum 9.7±0.3 nm, M. pusilla 8.4±0.6 nm and P articulosus 9.2±0.5 nm. Finally, to ascertain the role of fimbriae in host-mycoparasite interactions, AU was incubated with P. virginiana and M. pusilla (mycoparasite/susceptible host) and with P. virginiana and P . articulosus (mycoparasite/ resistant host). It was observed that AU decreased significantly the level of contact between P. virginiana and M. pusilla and between P. virginiana and P. articulosus in comparison to prelmmune serum treatments. Thus, it was proposed that fimbriae might play recognition and attachment roles in early events of mycoparasitism.